Otitis media (OM) is the most common childhood bacterial infection and the leading cause of conductive hearing loss in children. Overproduction of mucin, a hallmark of OM, plays an important role in the development of conductive hearing loss. However, little is known about the molecular mechanisms underlying mucin over production. The long-term objective is to understand the molecular mechanisms by which mucin is up regulated in OM so that the potential therapeutic targets can be identified for the inhibition of mucus overproduction in patients with OM. During the previous grant period, we have identified p38 MAPK as a key-signaling molecule mediating up-regulation of MUC5A C mucin by NTHi. Given the fact that approximately 40% of the episodes of OM are caused by S. pneumoniae, it is therefore important for us to investigate if p38 MAPK also plays a critical role in mediating up regulation of MUC5AC mucin in S. pneumoniae-induced OM. Interestingly, our preliminary studies indicate that S. pneumoniae up-regulates MUC5AC mucin via activation of MAPK ERK but not p38 signaling pathway, thereby supporting our hypothesis that Gram-positive S. pneumoniae, unlike Gram-negative NTHi, up-regulates MUC5AC mucin transcription via distinct signaling networks involving MAPK ERK but not p38 in the pathogenesis of OM. These interesting preliminary results have thus led us to fully investigate the molecular mechanisms underlying S. pneumoniae-induced up-regulation of MUC5AC mucin (short-term objective) towards identifying potential therapeutic targets for inhibiting mucin overproduction in OM. Aim 1. Determine if host receptor TLR4-dependent MyD88-IRAKTRAF6 signaling cascade is required for MUC5AC induction by S. pneumoniae by perturbing their signaling in vitro and in vivo. Aim 2, Determine ifRas-Raf-l-MEKl/2-ERKl/2 signaling pathway is required for MUC5AC induction by S. pneumoniae by perturbing they're signaling in vitro and in vivo. Aim 3. Determine ifMEKK3-JNKKl/2-JNKl/2 signaling pathway acts as a negative regulator for S. pneumoniae-induced MUC5AC transcription by perturbing their signaling in vitro and in vivo. Significance: The studies proposed in the current proposal together with the studies completed during the previous grant period will provide new insights into the molecular mechanisms underlying mucin overproduction in OM and lead to the identification of novel therapeutic targets for the inhibition of mucus overproduction in OM.